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Early elevation of cholesterol triggers atherosclerosis by altering macrophages

Irishu
Early elevation of cholesterol triggers atherosclerosis by altering macrophages

Discover how premature surges in cholesterol impair critical immune cells, increase the risk of heart disease, and highlight the urgency of proactive cholesterol management.

To work: Early intermittent hyperlipidemia alters tissue macrophages to fuel atherosclerosisImage Credit: crystal light / Shutterstock

In a recent study published in the journal NatureA group of researchers investigated how early intermittent hyperlipidemia (high blood fat levels) accelerates atherosclerosis (arterial plaque accumulation) by altering the number and homeostatic phenotype of resident tissue macrophages and identified the underlying biological mechanisms that contribute to increased risk of atherosclerotic cardiovascular disease (ASCVD).

Background

A large body of evidence links cholesterol to the development of atherosclerosis, in part through activation of the NLR Family Pyrin Domain-Containing 3 – Interleukin 1 Beta (NLRP3-IL1β) pathway.

However, the precise mechanisms driving inflammatory plaque formation in response to cholesterol overload remain unclear. Beyond Low Density Lipoprotein Cholesterol (LDL-C) levels, duration of exposure and area under the LDL-C-age curve are strong predictors of ASCVD events.

Cholesterol accumulation, especially in the early stages, confers a higher risk of CVD, and fluctuations in cholesterol levels, even with statin therapy, increase the risk of ASCVD.

Further research is needed to fully understand the precise mechanisms by which early and intermittent cholesterol changes accelerate atherosclerosis, particularly through alterations of actin filament organization and macrophage-associated pathways, enabling the development of more effective therapeutic strategies for ASCVD prevention.

About the work

Mice used in the study were of C57BL/6 (a common inbred strain of laboratory mice) background, including Lysozyme 2-Cre recombination heterozygous (Lyz2).Cre+/−), Low Density Lipoprotein Receptor knockout (Ldlr)−/−), Recombination Activating Gene 2 knockout (Rag2−/−) from Charles River and Apolipoprotein E knockout (Apoe)-/-) From Comparative Medicine.soft/soft mice were provided by researchers in Japan and Lyz2Cre+/− Neuropilin 1 floxed (Lyz2)Cre+/− Nrp1soft/soft) mice were bred at University College London. Apoe-/-lymphatic vessel endothelium hyaluronic acid receptor 1 Lyve1-Creative)/wt floxed colony stimulating factor 1 receptor (Csf1r)rsoft/soft Mice were generated by crossbreeding and some mice were injected with Adeno-Associated Virus serotype 8 encoding mutant Proprotein Convertase Subtilisin/Kexin Type 9 (D377Y variant) (AAV8-D377Y-mPCSK9) virus to induce hypercholesterolemia.

All in vivo experiments in mice were conducted under ethical approval from the relevant Institutional Review Boards, including protocol 2111-39587A of the Ministry of the Interior (UK), the Ethics Committee of the French National Institute for Health and Medical Research (INSERM) (France), the Institutional Animal Care and Use Committee (IACUC) (Singapore), and the United States of America (USA).

Mice were housed in pathogen-free facilities with controlled temperature and humidity. Atherosclerosis experiments generally began at 6 weeks of age, and mice were randomly assigned to experimental groups.

Sample sizes were determined to ensure adequate power to detect significant differences in lesion size. Bone marrow transplants involved irradiation of Ldlr−/− Mice were reconstituted with sex-matched donor bone marrow cells. Blood was collected to measure plasma cholesterol levels, and tissues were stained for histology and immunohistochemistry analysis of atherosclerotic lesions.

The study also used RNA sequencing and microbiota analysis to determine gene expression changes and the effect of gut microbiota on atherosclerosis. Ribonucleic Acid (RNA) sequencing and microbiota analysis were also conducted and gene expression was analyzed using RNAseq pipelines and related bioinformatics tools.

Study results

Animal models of atherosclerosis often rely on inducing hyperlipidemia to study disease progression, and plaque size is associated with circulating cholesterol levels.

Since the first model was developed, these models have mostly focused on creating long-term high cholesterol levels in animals late in life. However, these models do not account for lifelong changes in cholesterol exposure, which more closely mirrors the human experience.

To address this issue, a new model was developed to reveal early intermittent hyperlipidemia in mice and maintain overall cholesterol exposure (area under the curve) equivalent to traditional late persistent hyperlipidemia models.

In a series of experiments, Ldlr−/− Male mice were fed either late continuous Western diet (cWD) or early intermittent Western diet (iWD).

Plasma cholesterol levels and other physiological markers such as heart rate, weight, and blood pressure remained similar between the two groups. However, mice on the early intermittent diet had significantly larger atherosclerotic plaques than those on the continuous diet.

This result was consistent across both male and female mice, although the magnitude of the increase in plaque size varied more in females.

Plaques in iWD mice were not only larger but also showed increased inflammation and necrosis; plaques in iWD mice were characterized by increased inflammation, with greater numbers of macrophages and T cells and larger necrotic cores, suggesting altered plaque healing and increased disease severity.

The study also investigated the role of the gut microbiota, which can be affected by a Western diet and potentially influence the development of atherosclerosis.

After 6 weeks of diet, the composition of the gut microbiota differed slightly between the two groups. However, antibiotic treatment reduced but did not completely prevent the acceleration of atherosclerosis in iWD-fed mice, suggesting a limited role for the microbiota in this model.

Adaptive immunity was also investigated with Ldlr−/−/Rag2−/− Mice (deficient in T and B cells) still show accelerated atherosclerosis under iWD, suggesting that innate rather than adaptive immune responses are critical in this process.

Further analysis via RNA sequencing revealed significant changes in macrophage gene expression, particularly those associated with autophagy and efferocytosis, which are essential for plaque stability. These findings were linked to impaired function of resident-like macrophages, which are critical for maintaining arterial health.

Furthermore, RNA sequencing revealed that early intermittent hyperlipidemia reduced autophagy in macrophages, contributing to the formation of larger, more necrotic plaques observed in these mice.

Results

This study demonstrates that early intermittent hyperlipidemia is an important driver of atherosclerotic plaque development, even when cumulative cholesterol levels are similar to late hyperlipidemia.

Exposure to high cholesterol early in life significantly influences the progression of atherosclerosis, explaining the strong link between early cholesterol fluctuations and later cardiovascular events.

The study highlights the importance of controlling cholesterol levels early in life to prevent long-term cardiovascular risk. It identifies impaired autophagy and efferocytosis pathways in arterial macrophages, particularly resident-like macrophages, as major contributors to accelerated atherosclerosis. Early cholesterol control is vital to reduce long-term cardiovascular risk.